1. Field of the Invention
This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. In particular, the invention relates to microminiaturization of genetic, biochemical and bioanalytic processes. Specifically, the invention relates to performing bioanalytic and biotechnological processes that are dependent on incubating samples and solutions at different temperatures, and particularly reactions that required sequential sample incubation at alternating temperatures, including in vitro amplification reactions and most particularly the polymerase chain reaction. Methods for performing any of a wide variety of such microanalytical or microsynthetic processes using the microsystems apparatus of the invention are also provided.
2. Background of the Related Art
In the biological and biochemical arts, analytical procedures frequently require incubation of biological samples and reaction mixtures at temperatures greater than ambient temperature. Moreover, many bioanalytical and biosynthetic techniques require incubation at more than one temperature, either sequentially or over the course of a reaction scheme or protocol.
One example of such a bioanalytical reaction is the polymerase chain reaction. The polymerase chain reaction (PCR) is a technique that permits amplification and detection of nucleic acid sequences. See U.S. Pat. Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis. This technique has a wide variety of biological applications, including for example, DNA sequence analysis, probe generation, cloning of nucleic acid sequences, site-directed mutagenesis, detection of genetic mutations, diagnoses of viral infections, molecular “fingerprinting,” and the monitoring of contaminating microorganisms in biological fluids and other sources. The polymerase chain reaction comprises repeated rounds, or cycles, of target denaturation, primer annealing, and polymerase-mediated extension; the reaction process yields an exponential amplification of a specific target sequence.
Methods for miniaturizing and automating PCR are desirable in a wide variety of analytical contexts, particularly under conditions where a large multiplicity of samples must be analyzed simultaneously or when there is a small amount of sample to be analyzed.
In addition to PCR, other in vitro amplification procedures, including ligase chain reaction as disclosed in U.S. Pat. No. 4,988,617 to Landegren and Hood, are known and advantageously used in the prior art. More generally, several important methods known in the biotechnology arts, such as nucleic acid hybridization and sequencing, are dependent upon changing the temperature of solutions containing sample molecules in a controlled fashion. Automation and minimization of the performance of these methods are desirable goals in the art.
Mechanical and automated fluid handling systems and instruments produced to perform automated PCR have been disclosed in the prior art.
U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et al. teach fluid handling on microscale analytical devices.
International Application, Publication No. WO93/22053, published Nov. 11, 1993 to University of Pennsylvania disclose microfabricated detection structures.
International Application, Publication No. WO93/22058, published Nov. 11, 1993 to University of Pennsylvania disclose microfabricated structures for performing polynucleotide amplification.
Wilding et al., 1994, Clin. Chem. 40: 43-47 disclose manipulation of fluids on straight channels micromachined into silicon.
Kopp et al., 1998, Science 280: 1046 discloses microchips for performing in vitro amplification reactions using alternating regions of different temperature.
One drawback of the synthetic microchips disclosed in the prior art for performing PCR and other temperature-dependent bioanalytic reactions has been the difficulty in designing systems for moving fluids on the microchips through channels and reservoirs having diameter in the 10-100 μm range. This is due in part to the need for high-pressure pumping means for moving fluid through the small sizes of the components of these microchips. These disabilities of the prior art microchips limits the usefulness of these devices for miniaturizing and automating PCR and other bioanalytic processes.
Some of the present inventors have developed a microsystem platform and a micromanipulation device to manipulate said platform by rotation, thereby utilizing the centripetal forces resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform, as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; and 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.